拉曼光谱资料

拉曼光谱OVERVIEW1.Ramanspectragiveinformationonmolecularvibrationsandareobtainedfromchangesinthefrequencyoflightobservedinascatteringexperiment(inelasticscattering).2.Thephysicalpicturearisesfromconsideringchangesinpolarizability(induceddipolemoment)thatariseifavibrationoccursduringthetimetheelectronsareoscillatinginresponsetotheappliedradiation.3.Thegrossselectionruleisthatthevibrationalmotionmustproduceachangeinthepolarizabilityofthemolecule.4.Theanisotropyofthepolarizationofthescatteringcanbemeasured.Comparisonofthespectrapolarizedperpendicularandparalleltotheincidentradiationgivesinformationonthesymmetryofthevibrationalmotions.5.Ramanspectracanbeobtainedinwater.Thisisamajoradvantageoverinfraredspectra.6.ResonanceRamanspectraresultwhenthewavelengthoftheexcitinglightfallswithinanelectronicabsorptionbandofachromophoreinthemolecule.Somevibrationsassociatedwithsuchachromophoremaybeenhancedbyfactorsof1000ormore.7.Theexperimentalparametersofabandinaspectrumareitsposition()(whichisindependentofthefrequencyoftheexcitinglight),itsintensity(whichisdirectlyproportionaltoconcentration),anditspolarization.8.ThemainbiologicalapplicationsofconventionalRamanareverysimilartothoseforinfrared.ResonanceRamanaffordsameansofprobingselectivesitesinmolecules.Forexample,inmetalloproteins,Ramancangiveinformationonthenatureoftheliganddirectlyattachedtothemetal.6.1引言拉曼光谱和红外光谱都反映了分子振动的信息，但其原理却有很大差别：红外光谱是吸收光谱，而拉曼光谱是散射光谱。红外光谱的信息是从分子对入射电磁波的吸收得到的，而拉曼光谱的信息是从入射光与散射光频率的差别得到的。拉曼光谱的突出优点是可以很容易地测量含水的样品，而且拉曼散射光可以在紫外和可见光波段量测。由于紫外光和可见光能量很强，因此其量测比红外波段要容易和优越得多。拉曼光谱得名于印度物理学家拉曼(Raman)。1928年，拉曼首先从实验观察到单色的入射光投射到物质中后产生的散射，通过对散射光进行谱分析，首先发现散射光除了含有与入射光相同频率的光外，还包含有与入射光频率不同的光。以后人们将这种散射光与入射光频率不同的现象称为拉曼散射。拉曼因此获得诺贝尔奖。当一束入射光通过样品时，在各个方向上都发生散射。拉曼光谱仪收集和检测与入射光成直角的散射光。由于收集和检测的散射光强度非常低，因此拉曼光谱的应用和发展受到很大限制。六十年代激光开始广泛应用，拉曼光谱仪以激光作光源，光的单色性和强度都大大提高，拉曼散射仪的信号强度因而大大提高，拉曼光谱技术得以迅速发展，应用领域遍及物理，材料，化学，生物等学科，并已成为光谱学的一个分支拉曼光谱学。6.2拉曼光谱原理6.2.1光的散射：入射光通过样品后，除了被吸收的光之外，大部分沿入射方向穿过样品，一小部分光则改变方向，发生散射。一部分散射光的波长与入射光波长相同，这种散射称为瑞利散射(Rayleighscattering)。1899年，瑞利从实验中得出结论：晴天时天空呈兰色的原因是大气分子对阳光的散射。瑞利还证实：散射光的强度与波长的四次方成反比。这就是瑞利散射定律。由于组成白光的各种颜色的光中，兰光的波长最短，因而散射光强度最大。天空因而呈现兰色。瑞利当时并没有考虑到散射光的频率变化。他认为散射光与入射光的频率是相同的。所以后来把与入射光波长相同的散射称为瑞利散射，而把波长与入射光不同的散射称为拉曼散射。6.2.2拉曼散射的产生机械力学的解释光由光子组成，这是光的微粒性。光子与样品分子间的相互作用，可以用光子与样品分子之间的碰撞来解释。光照射样品时，光子和样品分子之间发生碰撞。如果碰撞时只是运动方向改变而未发生能量交换即发生了弹性碰撞，则光子的能量不变。由E=h，能量不变频率也就不变。这就是瑞利散射产生的原因。如果光子和样品分子间发生非弹性碰撞，即光子除改变运动方向外还有能量的改变，一部分能量碰撞时在光子和样品之间发生交换，光子的能量有所增减，则光的频率发生改变。从能级之间的跃迁来分析光子和样品分子之间的作用也可以从能级之间的跃迁来分析。Figure9.1Processesleadingtonormal,preresonance,andresonanceRamanscattering.(Forcomparison,theprocessesinvolvedinIRandfluorescenceareshown.)Thehorizontallinesrepresentdifferentvibrationalenergylevelsinthetwoelectronicstates.TheRamanscatteringspectrumisalsoindicated.NotethattheintensityoftheStokeslinesisgreaterthanthatoftheanti-StokesP240Fig9.1样品分子处于电子能级和振动能级的基态，入射光子的能量远大于振动能级跃迁所需要的能量，但又不足以将分子激发到电子能级激发态。这样，样品分子吸收光子后到达一种准激发状态，又称为虚能态。样品分子在准激发态时是不稳定的，它将回到电子能级的基态。若分子回到电子能级基态中的振动能级基态，则光子的能量未发生改变，发生瑞利散射。如果样品分子回到电子能级基态中的较高振动能级即某些振动激发态，则散射的光子能量小于入射光子的能量，其波长大于入射光。这时散射光谱的瑞利散射谱线较低频率侧将出现一根拉曼散射光的谱线，称为Stokes线。如果样品分子在与入射光子作用前的瞬间不是处于电子能级基态的最低振动能级，而是处于电子能级基态中的某个振动能级激发态，则入射光光子作用使之跃迁到准激发态后，该分子退激回到电子能级基态的振动能级基态，这样散射光能量大于入射光子能量，其谱线位于瑞利谱线的高频侧，称为anti-Stokes线。Stokes线和anti-Stokes线位于瑞利谱线两侧，间距相等，如图9.1所示。Stokes线和anti-Stokes线统称为拉曼谱线。由于振动能级间距还是比较大的，因此，根据波尔兹曼定律，在室温下，分子绝大多数处于振动能级基态，所以Stokes线的强度远远强于anti-Stokes线。拉曼光谱仪一般记录的都只是Stokes线。P241WorkedExample9.1OnlyStokesLinesAreUsuallyMeasuredExperimentallyUseFigure9.1tohelpexplainthestatementinthetitleoftheproblem.SolutionAnti-Stokeslinesinvolvetransitionsfromexcitedvibrationalstatesofthesamples.Thepopulationsofthesestatesareverymuchlessthanthepopulationofthegroundstate(seeChapter2).Fewermoleculesarethereforeavailabletogiveupaquantumofvibrationalenergy;thus，itiscustomarytomeasuretheStokeslinesinRamanexperiments.从光的波动性来分析由于光同时具有波动性，因此也可以从光的波动性分析拉曼散射的产生：电磁波的交变电场可以用E=E0cos(2't)表示，其中E是任意时刻t的电场强度，E0为交变电场的振幅，'为频率。样品分子的电荷分布在交变电场的作用下会发生变形，其正电荷和负电荷的中心会发生位置上的相对移动或分离，产生诱导偶极矩μ，μ=E，其中E为入射光的交变电场强度，是分子的极化率(polarizability)。分子极化率是衡量分子在电场作用下电荷分布发生改变的难易程度或诱导偶极矩(induceddipolemoment)的大小，也就是单位电场强度诱导产生的偶极矩的大小。如果分子的振动引起分子极化率的改变，则分子具有拉曼活性。以双原子分子为例，设分子极化率随分子振动而变化，则可按台劳级数展开。忽略高次项，可得到：=0+(d/dq)0q式中0是分子在平衡位置时的极化率，q=rre，是双原子分子核间距r与平衡位置时核间距r0的差。(d/dq)0表示平衡位置上对q的导数。由=E=[0+(d/dq)0q]E0cos(2't)根据前面红外原理中所推得的方程d2q/dt2=kq/的解q=q0cos2t可以有=[0+(d/dq)0q0cos2t]E0cos2't=0E0cos2't+q0E0(d/dq)0cos2tcos2't=0E0cos2't+(1/2)q0E0(d/dq)0[cos2('+)t+cos2(')t]式中前一项0E0cos2't对应于样品分子产生的波长未变化的散射即瑞利散射，第二项反映分子极化率随分子振动而改变(即(d/dq)0不为零)时分子产生的与入射光频率不同的散射光。散射光与入射光频率的差值即分子的振动频率，这就是拉曼散射。红外吸收的光频率是分子的振动频率，拉曼散射光与入射光的频率差也反映了分子振动能级之间的差。6.2.3拉曼散射的选择定则（参考书P242）外加交变电磁场作用于分子内的原子核和核外电子，可以使分子电荷分布的形状发生畸变，产生诱导偶极矩。极化率是分子在外加交变电磁场作用下产生诱导偶极矩大小的一种度量。极化率高，表明分子电荷分布容易发生变化。=E如果分子的振动过程中分子极化率也发生变化，则分子能对电磁波产生拉曼散射，称分子有拉曼活性。有红外活性的分子振动过程中有偶极矩的变化，而有拉曼活性的分子振动时伴随着分子极化率的改变。因此，具有固有偶极矩的极化基团，一般有明显的红外活性，而非极化基团没有明显的红外活性。拉曼光谱恰恰与红外光谱具有互补性。凡是具有对称中心的分子或基团，如果有红外活性，则没有拉曼活性;反之，如果没有红外活性，则拉曼活性比较明显。(Thisisanotherimportantprincipleofvibrationalspectroscopy,theruleofmutualexclusion.Thisrulestatesthatforanymoleculecontainingatrueinversioncenterofsymmetry,theinfraredactivevibrationsareRamaninactiveandviceversa.)一般分子或基团多数是没有对称中心的，因而很多基团常常同时具有红外和拉曼活性。当然，具体到某个基团的某个振动，红外活性和拉曼活性强弱可能有所不同。有的基团如乙烯分子的扭曲振动，则既无红外活性又无拉曼活性。下图显示了甲苯的红外谱和拉曼谱。可以看到：在某些频率处两者是吻合的，而在另一些频率上，只有一种谱上有峰。

FIGURE1.12.ComparisonofIR(top)andRaman(bottom)spectraoftoluene.SomelinesappearatthesamefrequencyinboththeIRandtheRamanspectrum.However,somelinesshowintheIRbutnotintheRamanspectrum.TheintensitiesoftheIRlinesarcdifferentfromthoseoftheRamanlines,althoughmanyofthemappearatthesamefrequency.Thisreflectsthedifferenceinselectivityoftwofundamentallydifferentprocesses.例.包含两个相同原子的双原子分子的红外活性和拉曼活性如何?(参考书P242例9.2)答：具有红外活性的分子振动必须引起分子固有偶极矩的变化。对于含两个相同原子的分子来说，由于它没有固有偶极矩，因此这个振动不可能发生固有偶极矩的变化，这个振动没有红外活性。但是在分子振动过程中，分子会变形，这就会引起电子相对于核的分布的变化。分子的极化率会反映这种变形，因而在分子振动中极化率会发生变化，这种振动因此有拉曼活性。例.CO2的拉曼光谱(参考书P242例9.3)。Figure9.2CO2有四个基本的振动模式，但其中只有一个有拉曼活性，为什麽?答：二氧化碳的对称伸缩振动改变了核和电子的相对位置，因而分子的极化率也发生改变，分子具有拉曼活性。在不对称伸缩振动中，一个键的伸展效果被另一个键的收缩抵消了，因此极化率总体上没有变化。另外两种振动模式也是如此。这里要注意的是：具有拉曼活性的振动是无红外活性的。事实上，如果一个分子具有对称中心，则具有红外活性的振动没有拉曼活性。反之，具有拉曼活性的振动没有红外活性。6.2拉曼谱的量测及参数RAMANINSTRUMENTATION6.2.1拉曼谱仪：TypesofInstrumentationandTrendsinthe1990sRamaninstrumentationhasaninterestingheritage,andinsomewaysthetechniquehasseenmorechanges,andmorediversitythananyotheranalyticaltechnique.Thismaybeduetotwoaspectsofitsevolutiononetechnicalandtheotherperceptionversusacceptance.TheRamaneffectisextremelyweak,andRamanspectroscopyhasobviouslygainedovertheyearsbytechnologythatimprovessignalanddetectability.Notallthesetechnological"breakthroughs"followthesamedevelopmentaltrail.Secondly,thetechniqueisinsomewaystoocloselyrelatedtoinfraredspectroscopy,andistreatedasapoorrelative.Ramanspectroscopy,inpracticaltermsandforspecificapplications,canbedemonstratedtohaveconsiderableadvantagesoverinfraredspectroscopy.However,asageneralanalyticaltechnique,itisdifficulttodemonstratethatitoffersanynetadvantages.And,ithasbeenseentohavesomecleardisadvantages,inparticularthecommoninterferencefrombroad-bandsamplefluorescence,whichcantotallymaskthespectrum.Consequently,whenpeoplejustifythepurchaseofnewlaboratoryinstrumentation,thesafedecisiontendstobeinfraredspectroscopy,whichiswellestablished,andhasevolvedconsistentlyoverthepast40+years.WithRaman,thereislesspracticalhistory,andtheinstrumentplatformsareconstantlychanging.Today,thereareacoupleoftechnologychoiceswhicharenotnecessarilymutuallyexclusivethereareprosandconstotheselection.Whenitcomestoaspecificapplication,however,itcanbeeasytodemonstratewhetherRamanorinfraredisthebetterchoice.TherearecurrentlytwomaintechnologicalapproachestothedesignofRamaninstrumentationamonochromatororspectrograph-basedCCDsystemandFT-Raman.Asmentionedabove,becauseofpracticalandtechnicalconstraints,thesetwoapproachesdonotnecessarilyproduceexactlythesameendresult.Qualitatively,bothgeneratethesamefundamentalspectrumforagivenmaterial,however,theoverallappearanceofthespectrumortheimpactofthesample,maybedifferent.Thissectionwillprovideanoverviewofinstrumentation asitexistsinthe1990s,anditwillprovideageneraldiscussionofthetrendsandapplications.ThegreatesttechnologicaldifficultyforRamanspectroscopyhasbeentheweaknessoftheRamanspectralsignal,comparedtothemagnitudeofthemainexcitationwavelength.TheintensityofaparticularRamanlinecanbeintherange10-6to10-10ofthemainexcitationline(possiblyevenaslowas10-12).Theissuesare:howtomeasuresuchalowsignalinthepresenceofadominantsignal,howtoremoveeffectivelytheinterferencefromthedominantsignal,and,ifpossiblehowtoenhancetheweakersignal.Oneapproachtoincreasingtheabsoluteintensityofthesignalistoincreasethepowerofthesourcethelaserpower.Whilethismaybepossible,itdoesnotremovethefundamentaldynamicrangeproblem,ortheinterferenceproblem,whichonlybecomesworsewithincreasedlaserpower.Foryears,thetraditionalRamaninstrumentsfeaturedscanningmonochromators.Theimportantcriteriaforthemonochromatordesignweretominimizestraylight,toenabletheveryweaksignalstobemeasured,andtodesignformaximumRayleighlinerejection.Theoriginalsolutionwastousemorethanonemonochromator,withcommercialsystemsbeingbasedondoubleandtriplemonochromators.Whilethesehadthedesiredopticalproperties,theuseofuptothreemonochromatorsmadetheseinstrumentsmechanicallycomplex,andseverelylimitedtheopticalthroughputoftheinstrument(downto5%orlessoverallefficiency).典型的拉曼谱仪装置图如下图所示。(A.T.Tu,FIGURE2.5.)EssentialpartsofaRamanspectrometer.Normallythescatteredlightat90isexamined.(instfig.pcx)由于散射效率大约为10-6~10-7，所以通常使用激光器作为光源，通过滤片和聚焦镜投射到样品上。这时光向各个方向散射。散射光包括瑞利散射(弹性散射)和拉曼散射(非弹性散射)。弹性光散射强度比拉曼散射高出103以上，所以色散系统必须精心设计，消除种种杂散光。为了达到高分辨率，一般采用双联或三联单色仪作为分光系统。一般在与入射光成90度的方向上接收散射光，采用光电倍增管作为接收器，然后经过信号处理电子系统和计算机，从显示屏或记录仪输出。输出参数为各个波数上的散射光绝对值，散射光波数的绝对值和拉曼波数(即入射光与散射光的波数差)，可以直接显示在屏幕上。Withtheintroductionoflaserlinerejectionfilters,andinparticulartheholographicnotchfilters,theneedforthesecondandthirdmonochromatorswaseffectivelyeliminated.Asidebenefitoftheuseoftheserejectionfiltersandtheeliminationoftheadditionalmonochromators,wastheassociatedreductionininstrumentsize. Thenextsignificanttechnologyboostwasprovidedbythemovetowardsdetectorarraysinitiallywithintensifiedphotodiodearrays,andmorerecentlywiththecooledCCD(charge-coupleddevice)arrays.Withsuchdevices,theneedtoscanthemonochromatormechanicallyisremoved.Theresultisahighefficiencyspectrographicsystemwithnomovingparts.Today,thelimitationsofthetechnologyarethecostofhigh-performancespectroscopicCCDarraycameras,andtheoverheadassociatedwithcooling.However,withmajoradvancesinimagingtechnologiesinthe1990stherehasbeenacorrespondingexpansioninCCDtechnology.Asaresult,itisanticipatedthatCCDdeviceswithimprovedperformanceandlowercostswillcontinuetobecomeavailable.ThetradesthathavetobemadewithaCCDdevicearespectralrangeversusspectralbandwidth,andtheimpactofthesignalcut-offbetween1000and1100nmforsilicon.Theseissueswillbediscussedingreaterdetail,later.WiththegainsinperformanceexperiencedwithFTIRinstrumentationcomparedtodispersiveinfraredinstruments,therehasbeenanaturaldesiretodeterminewhetherornotthesamelevelofperformancecanbeachievedforRamanspectroscopy.Originally,thisexperimentwasconsideredtobeimpracticalbecauseofnoiseconsiderationsandtheextraordinarilylargedynamicrangeinvolvedbetweentheexcitation(Rayleigh)lineandtheRamansignals.However,withtheadventofthelaserlinerejectionfiltersmentionedabove,HirschfeldandChasedemonstratedthefeasibilityofFT-Ramanspectroscopy.Originalexperimentswereperformedwiththe647.1nmlineofaKryptongaslaser,andwithasilicondetector.However,therealjustificationforthemovetoFT-Ramanspectroscopywastheabilitytousenear-infraredlasers.MovingfromvisibletoNIRexcitationhelpedtoremoveoneofthemajorinterferencesencounteredwithRamanspectroscopytheoccurrenceofbroadbandfluorescence.This,plustheexpectedgaininperformancefromthemultiplexadvantage,offeredbyFourierspectroscopy,madethetechniqueofFT-Ramananattractivealternativetotheconventionaldispersive-basedmethodsofmeasurement.Asecond,practicaladvantageforauseristhatRamancanbeperformedonanexistingFTIRinstrument,withoutsignificantredesign.Infact,mostofthemajorFTIRvendorsofferRamanasanaccessoryfortheirhigh-endinstruments.Inmostcases,the1064nmlineoftheNd:YAGlaser,operatingwithpowersofupto4W,areusedforexcitation.FollowingthesuccessoftheFT-Ramanaccessories,somededicatedFT-Ramansinstrumentswereproduced,withnotablegainsinperformancelinkedtotheoptimizationoftheopticalsystem.Inparticular,gainswereexperiencedbytheuseofhighreflectivityoptics,thereductioninthenumberofopticalelements,andtheuseofhighsensitivitydetectors,matchedtothelaserimage.Figure22SchematicdiagramofaFT-RamanSpectrometerOneofthemajorapplicationsofRamanspectroscopyhasbeenmicroscopy,withthebenefitsofthespatialresolutionofthelaserlightsource.Ramanmicroscopygainedpopularityinthemid1970sfromthepioneeringworkofDelhaye,etal.andbytheintroductionoftheMOLEbyInstrumentsSA.Later,followingthegaininpopularityofinfraredmicroscopy,withmicroscopeaccessoriesoptimizedforcommercialFTIRinstruments,aparallelimplementationwasmadeforFT-Raman.Likewise,commercialRamanmicroscopesareofferedeitherasaccessoriesorasdedicatedsystemsforusewithCCD-basedtechnology.AnimportantrecentdevelopmentisintheareaofRamanspectroscopicimagingmicroscopy,atwo-dimensionalexperiment,wheretheRamanspectrumisscannedwithanAOTFdevice,andthemainimageisgeneratedbyaCCDarray.Thistechnologyisexpectedtohavesignificantimpactonstudiesinmaterialsscience,inpolymerchemistry,andintheareaofbiologicalandmedicalresearch.OneofthemajorbenefitsofRamanspectroscopyisthefactthattheprimarymeasurementinvolvesvisibleorNIRradiation.Thispermitstheuseofconventionalglassorquartzopticsforimaging.Furthermore,itopensuptheopportunitytouseopticalfibersforremotesampling.Insuchanarrangement,asinglefiberisusedforthetransmissionoflaserradiationtothesample,andsecondfiber,oraseriesoffibers(Figure19),transmitstheRamanscatteredradiationbacktothespectrometer.Figure19SchematicdiagramofaRamanfiber-opticsamplingprobefeaturingafiber-opticBundleTheconstructionofthesample-lightinterfaceinthiscaseisveryimportanttomaximizethecouplingbetweenthelaserandthesample,andthesubsequentcollectionofthescatteredradiation.Silicafibersarenormallyused,whichcantransmitvisibleornearinfraredradiationoverrelativelylongdistanceswithoutsignificantlightloss.Usually,theRamanspectrumfromsilicaisveryweak,however,overthedistancecoveredbyopticalfibers,thecontributioncanbesignificant.Toovercomethisproblem,samplingprobesfeaturingopticalfilteringinthemeasurementheadareutilized.AnexampleofsuchaprobeheadisshowninFigure20.Figure20SchematicdiagramofaRamanfiber-opticsamplingprobefeaturingfilterelementsinthemeasurementhead(CourtesyofKaiserOpticalSystems,Inc.)Asnotedearlier,thereareseveralimportantareasofapplicationwhereRamanexcelsoverinfrared.Often,thesearebasedonpracticalissues,suchastheabilitytouseglassintheopticalsystem,thelowerRamanscatteringofwater,whichpermitsthestudyofaqueousmedia,andtheopportunitytohavenoncontactsampling.Asecondaryissueisthatunlikemid-infraredspectroscopy,thereisnointerferencefromatmosphericwatervapororcarbondioxide.This,coupledtothescalingdownininstrumentsize,theabilitytoperformremotemeasurements,andtheavailabilityofmechanicallysimpleinstruments,hasmadeRamanapracticaltoolforprocessapplications.Applicationshaverangedfromrawmaterialscreeningtoreactionmonitoring,withamajorfocusontheanalysisofpolymericproducts.6.2.2.拉曼谱参数：(参考书P243－245)拉曼谱的参数主要是谱峰的位置和强度。谱峰(谱线)位置：峰位是样品分子电子能级基态的振动态性质的一种反映。它是用入射光与散射光的波数差来表示的。峰位的移动与激发光的频率无关强度：Unlikethetraditionalinfraredmeasurement,theRamaneffectisanemissionphenomenon,andisnotconstrainedbythelawsofabsorption.TheintensityofarecordedspectralfeatureisalinearfunctionofthecontributionoftheRamanscatteringcenter,andtheintensityoftheincidentlightsource.Themeasuredintensityfunctionisnotconstantacrossaspectrum,andisconstrainedbytheresponseofthedetectorattheabsolutefrequency(wavelength)ofthepointofmeasurement.Also,Ramanscatteringisnotalineareffect,andvariesasafunctionof4,whereistheabsolutewavelengthofthescatteredradiation.WhileRamanintensitymaybeusedanalyticallytomeasuretheconcentrationofananalyte,itisnecessarytostandardizetheoutputintermsoftheincidentlightintensity,thedetectorresponseandtheRamanscatteringterm,bothasafunctionoftheabsolutemeasuredwavelength.拉曼散射强度与产生谱线的特定物质的浓度有关，成正比例关系。而在红外谱中，谱的强度与样品浓度成指数关系。)样品分子量也与拉曼散射有关，样品分子量增加，拉曼散射强度一般也会增加。对于一定的样品，强度I与入射光强度I0、散射光频率s、分子极化率有如下关系：I=CI0s42这里C是一个常数。在共振拉曼谱中，谱的加强是由于极化率的增加引起的。退偏比(depolarizationratio)如参考书P242中所说，一个分子的电荷在某一个方向上可能比在另一个方向上容易变形，称这种分子的极化率各向异性；相反，则称为各向同性。把一个朝向与偏振光平行的偏振片放在偏振光的路径上，则偏振光可以通过偏振片。若偏振片转动90度，则偏振光不能通过偏振片。如果一个分子位于原点0，对入射光进行散射。下图只表示了沿Y方向散射的射向观测者的光。对于高度对称的分子如CH4和SF6而言，极化率是各向同性的。当这类分子的完全对称的振动模式(例如CH4中C-H键的伸缩振动)与在XZ平面上偏振的入射光相互作用时，散射光将在YZ平面上偏振。这样，当一个偏振片平行于YZ平面放置时，只有这个平面上的散射光能够通过。这部分光的强度称为I∥。若偏振片转动90度，则YZ平面上的光将无法通过。偏振片处于这个位置时测得的散射光强度称为I⊥。=I⊥/I∥定义为退偏比。对于CH4的对称C-H伸缩振动来说，退偏比=0。

(A.T.TuFIGURE1.32.)Theintensityofscatteredtightcanbemeasuredtwodifferentways.(A)Lightcomesthroughaparallel-orientedpolarizerandisparalleltotheincidentlight(I).(B)Lightcomesthroughaperpendicularlyorientedpolarizerandisperpendiculartotheincidentlight(I).TheratioofItoIiscalledthedepolarizationratio,anditisrelatedtosymmetryofvibrationalmodes.大多数分子的对称程度比CH4或SF6小，因此，极化率是各向异性的。一般来说，分子散射的光在XZ和ZY平面上都有偏振。在平行于入射光偏振方向的方向上(如图中的Z方向)，测到的散射光强度与垂直于入射光偏振方向的方向(如图中所示的X方向)上测得的强度是不同的，但其比值一般不为零。 如果入射光是平面偏振光，则退偏比=I⊥/I∥=32a/(45i+42a)其中i是极化率的各向同性部分，a是极化率的各向异性部分，I⊥是垂直于入射光的方向上偏振的散射光强度，I∥是平行于入射光的方向上偏振的散射光强度。 对于平面偏振光来说，退偏比与振动的不对称程度有关，其值在0到3/4之间。任何分子的不完全对称的振动，其退偏比为3/4(i=0)。对于完全对称的振动，≤3/4。上面提到的CH4的例子中，退偏比为。但一般来说，值在0到3/4之间，其大小取决于分子极化性质的变化和分子键的对称性。一个完全对称的振动在进行任何对称操作后不变，这些对称操作只是交换了分子中对等原子的平衡位置。因此，退偏比的量测可以提供有关分子对称性的信息，而且有助于拉曼谱线的指认。6.2.3增强拉曼光谱：TherearesomeenhancedRamanmethods,whichforsomecompoundsproducesignificantlyintensifiedRamanspectra,andwhichovercomesomeaspectsofthisdichotomy.TwosuchtechniquesareresonanceRaman（参考书P243）andsurface-enhancedRamanspectroscopy(SERS).OneotherRaman-basedtechniqueworthyofmentioniscoherentanti-StokesRamanspectroscopy(CARS)furtherdiscussionofthistechniqueisbeyondthescopeofthisbook.ResonanceRamanisparticularlyinterestingbecauseitcanturnRamanintoahighlyspecificprobeforcertainfunctionalgroupsorchemicalsites.ResonanceRamanoccurswhenthelaserexcitationfrequencycoincideswithanelectronicabsorptionband.Inthiscase,thevibrationsassociatedwiththeabsorbingchromophoreareenhancedbyasmuchas103to106timesthenormalRamanintensity.TheseintensifiedRamanlinesarelinkedtothespecificchromophoresiteandfunctionalgroupsorsites,withinthemolecule,thatinteractwiththechromophoricgroup.TheearlyresonanceRamanexperimentswerewiththevisiblelinesoftheargonionlaser,andthisobviouslyconstrainedthetechniquetoalimitedsetofcoloredcompounds.Ofthese,theworkwiththehemechromophoreofthehemeglobinmoleculewerethemostsignificant.Itispossibletoobservetheinfluenceofexternalmolecularligands,suchasoxygen,carbonmonoxideandcyanide,onthecriticalhemesite,freeofinterferencefromtheremainingoftheproteinstructures.Movingtoshorterwavelengthsfromthevisibletowards,andintotheultravioletregionsmight,atfirstsite,seemtobeimpracticalbecauseonenormallyequateshighlevelsofnativefluorescencewiththeuseofUVexcitation.However,manycompoundsabsorbintheultravioletspectralregion,andsothereisahighprobabilityfortheresonanceRamaneffecttooccur.Also,ithasbeenobservedthatbelow260nmexcitationthatthereisvirtuallynointerferencefromfluorescence.Oneofthemainissuesherehasbeentheappropriateselectionofa laseroperatingintheUVrange.OneapproachistouseadyelaserpumpedbyaNd:YAGoraXeCIexcimerlaser,coupledtofrequencydoublingandtriplingcrystalstoprovideawavelengthselectablerangeof200to750nm(Nd:YAG)and206-950nm(excimer).Boththeselasersystemsprovideapulsedlaseroutput.Apracticalalternative,wherecontinuouswavelengthtuningisnotarequirement,isanintracavityfrequencydoubledargonionlaserwhichprovidescontinuousoutputoffiveexcitationlinesinrange230nmto260nm.当入射光引起分子中电荷的平移时，则发生散射。电荷的平移通过分子极化率反映出来。散射的强度与极化率的平方成正比。当激发光的频率接近且小于两个电子能级之间的频率差时，则产生所谓的preresonance，当激发光的频率等于两个电子能级之间的频率差时，则会发生共振，这时产生很大的电子电荷频移或分子变形的概率很高，这时就会产生对光的吸收。这也说明对于某些振动，当入射光接近或等于某个吸收跃迁频率时，极化率会变得比较大。这种振动称为共振加强的(resonanceenhanced)振动。这种加强取决于电子跃迁的强度及振动的对称性。如果只有一个单一的电子态，则振动加强必须是对称的。即这种振动不能改变分子的对称性。如果某个被激发的生色团有不止一个电子跃迁，则振动的对称性就不那么重要了。共振拉曼谱典型的增强倍数是102~103，因此共振拉曼谱在10-4moldm-3或更低的样品浓度下即可测得。这样，共振拉曼谱就提供了一种以接近紫外光谱的灵敏度选择性地探测生色团振动频率的手段。共振拉曼在研究生物大分子的结构和功能时很有用，多生物分子都含有能给出共振拉曼谱的基团，如类胡萝卜素、黄素、视紫红质、各种含铜与铁的化合物、叶绿素等。共振拉曼谱仪的使用范围主要受到激光光源频率有限这一现实情况的限制。 Asnoted,anothertechniquethatprovidesanenhancedRamanspectral outputisSERSsurface-enhancedRamanspectroscopy.UnlikeresonanceRaman,thelaserwavelengthisnotimportant,unlessaqueous-basedmeasurementsaremadewithnear-infraredexcitation.Asthenameimplies,themeasurementisspecificinnature,andsomewhatlimitedinapplicationtosurfacesorinterfaces.MoststudiesofSERShavebeenperformedonelectrodesurfaces.Ansignal enhancement,intheorderof106isobservedforadsorbedspeciesoncertainmetallicelectrodesurfaces,notablymetalssuchasgold,silver,platinumandto someextentcopper.Theoriginalexperimentsinvolvedcalomel(Hg2C12)ona mercurysurface,andlaterworkinvolvedorganics,suchaspyridineadsorbedonroughenedsilverelectrodesurfaces.Theenhancementphenomenonisnotrestrictedtoelectrodes,andsimilareffectshavebeenobservedforothersubstratesinvolvingmetals,suchascolloidalsuspensionsofmetalsandmetalsdepositedorembeddedinoxides.Acriticalfactorinalltheexperimentsisthesurfaceroughness,whichisnominallyattheatomicscale.Theoriginoftheenhancementisbelievedtobeassociatedwithanincreasedelectricfieldintheregionofthemoleculeunderstudy.Althoughexperimentsinvolvingchargedmetalsurfaces mightappeartobelimited,thephenomenondoesopenupinterestingapplications intheareaofmetalliccorrosion,thestudyofbatteriesmaterials,andnew electrolyticstudiesinthecontestedareaofcoldfusion.6.3拉曼光谱的特点和应用6.3.1.优缺点：AdvantagesandDisadvantagesofVibrationalSpectroscopyRamanandinfraredspectroscopyforproteinandnucleicacidstructureanalysishavethefollowingnotableadvantages:1.Ramanandinfraredspectroscopyarenondestructivetechniques.Ordinarilythesamplemayberecoveredandassayedforbiologicalactivityafterspectroscopicexamination.2.Ramanandinfraredmethodsareapplicabletosamplesofvirtuallyanymorphologicalform.Forproteinsandnucleicacids,thisincludessolutions(aqueousandnonaqueous),suspensions,precipitates,gels,films,fibers,singlecrystals,andpolycrystallineandamorphoussolids.Dataobtainedfromagivensampleinonemorphologicalstatearegenerallytransferabletoanothermorphologicalstateofthesamesample.Thishasimportantpracticalbenefitsforexample,incomparingthemolecularstructureofaproteininthecrystalwiththatprevailinginsolution.3.Asmallsamplevolumeisrequiredforthesemethods.Approximately1lissufficientforconventionalRamanspectroscopyandapproximately10lforFouriertransforminfraredspectroscopy.Thisrepresentsanadvantageovermanyotherstructuralmethods,includingX-raycrystallographyandmagneticresonancespectroscopy.4.Ramanscatteringandinfraredabsorptionprocessesoccuronatimescalethatisveryshort(1015sec)incomparisontothetimescalesoffluorescence(>109sec)andnuclearmagneticresonancephenomena((106sec).Thus,vibrationalspectroscopyissuitablefortime-resolvedstudiesofbiologicalprocessesthatareinaccessiblebyfluorescenceandmagneticresonancemethods.5.ThereexistsalargedatabaseofinfraredandRamanspectraofproteins,nucleicacids,andtheirconstituents,forwhichreliablebandassignments,normalmodeanalyses,andspectra-structurecorrelationshavebeenmade.Thisfacilitatesinterpretationoftheoftencomplexvibrationalspectraobtainedfromproteins,nucleicacids,andtheircomplexes.ThefollowingadvantagesarespecifictoRamanspectroscopy:1.BothH2OandD2OgenerateveryweakRamanscattering,thusproducingrelativelylittleinterferencewiththeRamanspectrumofthedissolvedsolute.Thisconstitutesasignificantad